Progressive multifocal leukoencephalopathy (PML) is a demyelinating disease of the central nervous system (CNS) resulting from the lytic infection of oligodendrocytes by the human polyomavirus, JC virus (PML was once considered a rare complication of middle-aged and elderly patients with lymphoprolifera diseases. In recent years, however, increasingly high incidence of PML in AIDS patients than those wit immunosuppressive disorders led us to believe that PML may also be regarded as a AIDS-associated disease. Lytic phase of JCV appears to be highly complex and remains elusive. A growing body of experimental evidence suggests that the regulation of JC viral gene expression and replication is not mediated solely by the presence absence of a particular transcription factor in a given cell. Rather delicate interactions among transcription or interactions between host and viral regulatory proteins appear to be important determining factors in respect. In support of this hypothesis, we have recently presented evidence of specific functional interactions between a cellular factor, YB-1, and JCV T-antigen and showed that both proteins play important roles in J expression. In addition, our most recent published and unpublished data indicate that JCV late Agno protein, antigen, also appears to have regulatory roles in both viral gene expression and DNA replication through interaction with viral and cellular proteins, notably with T-antigen and YB-1. Hence, here we propose investigate the molecular mechanisms involved in regulation of JCV by the functional interplay between T-antigen, Agno and YB-1. By employing molecular biology, genetics and virological approaches, we will i) perform thorough molecular virological studies to determine the role of Agno in regulation of JCV gene transcription and replication by examining its physical and functional interaction with T-antigen and YB- 1; ii) investigate the function importance of Agno viral lytic cycle by means of both ectopic expression of Agno early in infection mutational analysis and; iii) characterize the potential phosphorylation sites of Agno protein and investigate the role in JCV life cycle. The results from such comprehensive studies will provide valuable information underlying mechanisms involved in both JCV gene regulation and replication; and thereby the progression of JCV-induced CNS disease.